这项研究得到了澳大利亚 RMH 神经科学基金会的支持;澳大利亚大脑基金会;澳大利亚 RACP Thyne 里德奖学金;由认知科学和技术委员会的项目赠款, 伊朗对阿巴斯 Haghparast。

这里解释的所有程序都是按照《动物保育和使用指南》批准和执行的, 并经 Florey 研究所动物伦理委员会或神经科学研究中心批准。
1. 住房、搬运和食物限制
<ol><li>使用成年 (通常8周大) 雄性大鼠 (任何菌株), 并保持他们在房间内的12小时的光/暗循环。</li>
	<li>限制他们的食物接触, 鼓励动物执行任务。 注意: 建议动物的住房, 因为它提供更好的控制每一个人的食物摄入量。</li>
	<li>1-3 天, 每天处理动物大约5分钟, 2x。 注意: 处理熟悉与人类接触的动物, 并减少在整个实验动物的压力和焦虑水平。</li>
	<li>在每次处理会话后对动物进行称量。使用第一天的重量作为免费喂养量, 并保持动物约 80-85% 的免费喂养量。</li>
</ol>2. 实验设置
<ol><li>使用装有3个分区或武器的 T 迷宫、2个刺激扬声器、5个可伸缩门和5个红外运动传感器或红外光束 (IRB) 传感器。 注: T 型迷宫可由中密度纤维板 (MDF) 或聚氯乙烯 (PVC) 构成。</li>
	<li>控制在这里构造的迷宫由一个 Arduino 微控制器。</li>
	<li>将 Arduino 代码复制到计算机中。</li>
	<li>从软件网站下载 Arduino 软件并将其安装在计算机上。</li>
	<li>使用 USB 端口将微控制器连接到计算机。</li>
	<li>在 "计算机" 上, 单击 "软件" 图标, 转到 "工具", 然后选择 "端口"。</li>
	<li>从下拉菜单中, 选择将软件连接到计算机的 COM 端口 (通信端口)。</li>
	<li>转到工具并选择主板。从下拉菜单中, 选择控制 T 迷宫的 Arduino 的类型。</li>
	<li>单击 "界面" 窗口左上方的 "上载"。选择 Arduino 代码。等待进程完成。</li>
	<li>单击 "接口" 窗口右上的 "串行监视器"。然后, 在新的弹出窗口中, 将波特率更改为115200。</li>
</ol>3. 适应迷宫
<ol><li>在每一次的适应之前, 把动物带到实验室至少1小时之前的实验。</li>
	<li>在每个球门臂上留出10块糖丸, 让所有的门都开着。 注意: 在这个阶段, 没有必要跟踪动物的运动 (图 1)。</li>
	<li>天 1-3: 在迷宫中单独放置动物10分钟, 每天 1x, 探索迷宫没有限制。</li>
	<li>天 4-5: 安置动物单独地在迷宫。每只手臂留出2粒, 让他们从两边取样。将动物从迷宫中取出后, 立即从两侧或5分钟后在迷宫中进食。 注意: 这一阶段和上一阶段的主要区别是, 动物需要从迷宫中的两个球后立即被移除。这将使动物熟悉在试验结束时从迷宫中移除。</li>
	<li>每节课后, 用70% 乙醇清洁迷宫的地板, 并确保在将下一个动物放到迷宫中之前, 乙醇已经蒸发。</li>
</ol>4. 歧视培训
<ol><li>
迷宫准备
	<ol>
<li>6天: 实验前, 把动物带到实验室至少1小时。</li>
		<li>运行 Arduino 程序, 并将试验编号设置为 14, 其中前4项试验将被强制选择, 其余的将是选择试验。 注意: 该程序将随机生成相同数量的试验被分配到 T 迷宫的左和右端。</li>
		<li>在每次试验开始之前, 将4粒球放在目标臂中, 将被提示为 HRA, 另一臂是上帝抵抗军的2颗小球。</li>
	</ol>
</li>
	<li>
强迫选择试验训练 (4 项试验)
	<ol>
<li>伪随机阻断一只手臂在每次试验前使动物被迫选择另一只手臂。 注: 被阻断的手臂可以是一个高奖赏臂或低奖赏臂, 而听觉信号突出的侧面的 HRA。</li>
		<li>将动物放在 "开始" 框中 (图 1)。5-7 秒后, 同时打开启动门并在 Arduino 的界面窗口中单击 "开始"。</li>
		<li>进食小球后立即收集动物, 或在迷宫中5分钟后采集。</li>
		<li>把动物留在笼子里2分钟。</li>
	</ol>
</li>
	<li>
选择试用培训 (10 项试验)
	<ol>
<li>在每次试验之前, 关闭门 B 在胳膊被选择由软件是 HRA。离开门-在相反的手臂 (上帝抵抗军) 开放。</li>
		<li>将动物放在 "开始" 框中 (图 1)。5-7 秒后, 同时打开启动门并单击 "开始"。</li>
		<li>让动物自由选择任一臂。如果动物选择了 HRA, 打开门, 让动物进入房间, 关上门, 打开门 B 立即给动物进入选定的食物好。</li>
		<li>如果动物选择了上帝抵抗军, 打开门 B, 以使动物获得食物的良好。</li>
		<li>在吃完所选食品中的所有食物后, 将其移走, 并将其留在笼中2分钟。</li>
		<li>为每种动物完成10项选择试验, 并在每次试验中记录动物的选择 (HRA 或上帝抵抗军)。</li>
		<li>在每次训练后计算所有选择试验的高奖赏选择百分比。</li>
		<li>完成14步后, 每个红外线传感器获得的各个时间都显示在 Arduino 界面窗口中。</li>
	</ol>
</li>
</ol>5. 延迟培训
<ol><li>一旦每一个动物已经达到80% 的人权委员会在歧视训练 (步骤 4), 开始延迟训练, 每天运行10试验, 每一个动物。在这一阶段, 门 B 将被打开后, 只有5的延迟, 当动物选择 HRA。</li>
	<li>如果该动物在训练课程结束时达到80% 的人权委员会, 在5的延迟时间内, 将推迟到十年代进行下一个培训课程。</li>
	<li>一旦该动物已达到80% 的人权委员会, 十年代的延迟, 增加推迟到十五年代的下一个培训课程。</li>
	<li>记录每项试验的动物选择, 在每次训练后计算人权委员会。</li>
	<li>试验完成后, 每个红外线传感器所获得的各个时间都显示在 Arduino 界面窗口中。 注 1: 单击Enter时计时器开始。"Time_decision" 是鼻子戳到 IRB-1 的时间。"Time_left_1" 是动物打破 IRB-1L 和 ' Time_left_2 ' 的时候, IRB-2L 被打破了。如果将行为研究与电生理学或神经成像 (如钙成像) 方法结合起来, 则建议用胶片记录更好地将动物的行为与神经信号同步。</li>
</ol>6. 电生理学 (电极制造)
<ol><li>使用单极或双极性电极记录局部磁场电位 (LFPs)。 注意: 建议使用双极性电极, 因为它能使局部重新参照, 以减少体积传导的影响。因此, 记录的 LFP 是2电极之间的差电压与200微米距离之间的提示。</li>
	<li>要制造两极电极, 请将2个钨电极粘合在一起, 或将2不锈钢线连接在一起 (通常, 使用50微米直径的粉煤灰涂层不锈钢线)。</li>
	<li>将接地和记录线焊接到迷你插头/插座上, 成为电生理记录系统的连接器。</li>
</ol>7. 麻醉
<ol><li>要植入电极, 麻醉大鼠腹腔注射氯胺酮 (100 毫克/千克) 和甲苯噻嗪 (8 毫克/千克) 混合物, 或管理异氟醚, 如以下步骤所述。 注意: 建议使用异氟醚, 因为它的麻醉深度控制得到改善。</li>
	<li>使用有控制氧和麻醉蒸汽流的麻醉机。</li>
	<li>用异氟醚麻醉动物, 将其放置在感应腔内, 在麻醉机上将异氟醚设置为大约4% 和 500-1000 毫升/分钟的氧气。</li>
	<li>继续诱导直到动物的矫正反射消失。</li>
	<li>在机器上设置 1-2.5% 异氟醚和 300-400 毫升/分氧的麻醉维持率。</li>
	<li>将隔热垫 (棉绒) 放在立体定向装置上, 然后将该动物固定在立体定向装置中。</li>
	<li>通过捏动物的脚趾来测量麻醉的水平。</li>
</ol>8. 手术方法
<ol><li>使用电动剃须刀或剪刀, 剃掉动物的头部, 在切口是要作出。</li>
	<li>用乙醇擦洗手术区, 然后用 povidone–iodine, 4%。</li>
	<li>在手术中应用眼部润滑剂保护动物的角膜。</li>
	<li>使用纸巾钳, 轻轻地拉在动物的眼睛和耳朵之间的皮肤。使用剪刀, 切断钳夹之间的皮肤, 取出骨膜。</li>
	<li>使用过氧化氢清洁和消毒头骨。</li>
	<li>使用立体定位坐标找到电极插入点的位置, 并在头骨上标记一个永久性标记。</li>
	<li>使用手持钻头为接地电极制造一个孔, 一个用于锚固植入物, 每个记录电极上有一个孔。</li>
	<li>插入电极以非常缓慢的速度, 以尽量减少可能的皮质和皮层下的损害。</li>
	<li>用小螺丝将接地电极连接到头骨上。</li>
	<li>使用薄层的牙科丙烯酸水泥, 确保连接器插头/插座到位。</li>
	<li>注射丁丙诺啡 (15 µg/千克, 皮下) 或 meloxicam (1-3 毫克/千克, 皮下) 作为止痛药。</li>
	<li>把动物留在一个温暖的垫子上的笼子里恢复。至少每30分钟观察一次动物, 直到它从麻醉剂中恢复。</li>
</ol>9. 程序后培训
<ol><li>手术后, 至少允许10天的恢复期。将动物单独带到食物和水的广告随意并每天监视它们 (参见 Rudebeck、沃尔顿、史密斯、班诺曼和拉什沃思7 , 了解更多信息)。</li>
	<li>在恢复之后, 开始将动物的饮食限制在免费喂养量的85%。</li>
	<li>重新培训迷宫中的动物, 以召回歧视阶段 (步骤 4)。</li>
	<li>继续对老鼠进行重新训练, 直到他们达到80% 的人权委员会。</li>
</ol>

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作者没有什么可透露的。

啮齿目动物长期被使用了在处理不同的题目的神经科学研究研究, 从认知能力例如学习和记忆2,14和增强的行为7,15,16对器官的中央控制17,18和神经药理学19,20。拟议的议定书解释了一个复杂的行为?...

决策是根据决策者的价值和偏好以及所选行动1的后果来识别和选择选择的过程。尽管决策在不同领域 (即经济学、心理学和神经科学) 中得到了广泛的研究, 但这种认知能力背后的神经机制尚未得到充分的理解。决策的两个子类是感性决策和增强导向决策。虽然它们包含相当多的重叠元素和概念, 感性决策依赖于现有的感官信息1,2, 而加强导向决策处理相对价值在特定时间刻度3中获得的操作。增强决策的一个重要方面是成本效益分析, 它是通过计算给定选择的好处并减去每种备选方案1的相关成本而直观地执行的。
T 迷宫 (或变种 Y 迷宫) 是使用啮齿动物的认知实验中使用最广泛的迷宫之一。动物被放置在起始臂 (T 的底部), 并允许选择目标臂 (其中一个侧臂)。任务, 如强迫交替或左-右歧视主要是用来与啮齿类动物在 T 迷宫测试参考和工作记忆4。T 迷宫也广泛用于决策实验5,6,7。在最简单的设计中, 奖励只放在一个目标臂上。这种选择是可以预测的, 而无论奖励价值如何, 动物们肯定更喜欢奖赏而不是一无所有。另一种选择是在两个目标武器中放置奖励, 然后让动物根据几个参数 (即动物的自然喜好、奖励的价值和支付的费用) 选择哪条路径。在基于价值的设计中, 由于具有称量尺度属性, 任务更加复杂。这样, 动物通过在两种替代品之间选择, 以及在行动的成本 (即等待的量 (延迟) 或获得奖励所需的努力 (基于努力的工作量) 之间, 得到不同的价值奖赏,每个作出贡献的决定,5,6。
在传统的基于延迟的 T 迷宫决策中, 动物被训练来选择高奖赏臂 (HRA), 避免相反的低奖赏臂 (上帝军)。在整个实验中, HRA 和上帝抵抗军的两侧保持不变。尽管上述任务在文献中有很好的记载, 但它还是有几个程序上的弊端。首先, 通过有一个固定的目标手臂, 动物知道哪一个手臂选择从一开始的试验。在这种情况下, 动物可以根据记忆而不是决策来选择目标臂。因此, 在一个基于延迟的决策范式中, 如果动物因为研究干预而选择了低报酬, 就不清楚这是由于记忆力的丧失还是学习干预的缘故。记忆控制小组分离观察的行为从记忆问题可能被考虑, 但这负担研究员和动物一样由于另外的工作7。第二个问题是动物做出决定的时刻: 一旦动物到达决策区 (所有三臂的交界处), 他们通常会向左和向右看, 权衡每只手臂的成本和利益, 然后做出决定。然而, 经过几次试验, 他们在到达决策区之前进行这样的计算, 直接跑到奖励臂上。因此, 这两个缺点-一只手臂的预偏向, 并发现决策的时刻-都高度中断了对电生理和神经影像数据的解释。
在本文所解释的方法中, 首选臂 (HRA) 由听觉线索提示, 并可能因试用而异。动物通过进入测试区 (图 1) 启动试验, 并通过 "鼻子戳" 的红外线门触发听觉提示, 该闸门已放置在三臂的交界处。音频信号 (20 分贝, 介于500和 1000 ms) 是从扬声器在目标臂的末尾播放。

<table>
	<tbody>
		<tr>
			<td>T-maze</td>
			<td></td>
			<td></td>
			<td>Self made</td>
		</tr>
		<tr>
			<td>Dustless Precision Sugar Pellets</td>
			<td>TSE Systems Intl. Group</td>
			<td>F0023</td>
			<td>45 mg, Sucrose</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride Injection, USP</td>
			<td>Sigma-Aldrich</td>
			<td>6740-87-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma-Aldrich</td>
			<td>7361-61-7</td>
			<td></td>
		</tr>
		<tr>
			<td>stereotaxic device</td>
			<td>Stoelting</td>
			<td></td>
			<td></td>
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			<td>Isofluran</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-363629Rx</td>
			<td></td>
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			<td>PFA-coated stainless-steel wires</td>
			<td>A-M systems</td>
			<td></td>
			<td></td>
		</tr>
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			<td>acrylic cement</td>
			<td>Vertex, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(wooden or PVC (polyvinyl chloride)-made)</td>
			<td>local suppliers</td>
			<td></td>
			<td></td>
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			<td>Mini-Fit Power Connector</td>
			<td>Molex</td>
			<td>15243048</td>
			<td></td>
		</tr>
		<tr>
			<td>ethannol 70%</td>
			<td>Local suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>buprenorphine</td>
			<td>diamondback drugs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arduino UNO</td>
			<td>Arduino</td>
			<td></td>
			<td>https://www.arduino.cc/</td>
		</tr>
		<tr>
			<td>Infrared emitting diode</td>
			<td>Sharp</td>
			<td>GL480E00000F</td>
			<td>http://www.sharp-world.com/</td>
		</tr>
		<tr>
			<td>Chronux Toolbox</td>
			<td></td>
			<td></td>
			<td>Chronux.org</td>
		</tr>
		<tr>
			<td>Arduino codes</td>
			<td></td>
			<td></td>
			<td>https://github.com/dechuans/arduino-maze</td>
		</tr>
	</tbody>
</table>

operant protocols for assessing the cost-benefit analysis during reinforced decision making by rodents

强化导向决策是根据利益的相对价值及其后果选择竞争的行动路线之间的能力。这一过程是人类正常行为的组成部分, 被证明被神经和精神紊乱, 如成瘾、精神分裂症和抑郁症所扰乱。啮齿类动物早已被用来揭示人类认知的神经生物学。为此, 制定了若干行为任务;然而, 大多数是非自动化的, 是劳动密集型的。最近开发的开源微控制器使研究人员能够自动化基于操作的任务来评估各种认知任务, 规范刺激演示, 改进数据记录, 从而提高研究成果。在这里, 我们描述了一种自动化的基于延迟的增强导向决策任务, 使用由定制软件程序控制的操作性 T 迷宫。利用这些决策任务, 我们展示了大鼠前扣带皮层局部场电位活动的变化, 同时进行了基于延迟的成本和效益决策任务。

这里提供的数据是从左眶皮质 (LFP) 和前扣带皮质 (ACC) 的记录使用双极电极 (粉煤灰涂层不锈钢)。表 1显示了每个训练阶段的行为习得长度。目标位置的坐标是从鼠脑图谱9确定的, 如下: 为 AAC, 1.2 毫米前的 bregma, 0.8 毫米侧向中线, 和2毫米腹侧的头骨;对于 bregma, 3.5 毫米的前部, 2.3 毫米侧向中线, 5.4 毫米腹侧的头骨。
<p class="jove_content" fo:keep-...

Watch this Scientific Journal Video about Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents at JoVE.com

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

啮齿动物强化决策中成本效益分析的操作协议

成本效益分析是大脑在决策过程中进行的一种称重尺度方法。在这里, 我们提出了一个训练老鼠的协议, 在一个以操作为基础的决策范式中, 老鼠选择更高的奖赏, 代价是等待十五年代的接收。

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...

operant-protocols-for-assessing-cost-benefit-analysis-during

Research

JoVE Journal

Neuroscience

5.9K 次观看.  The University of Melbourne. 成本效益分析是大脑在决策过程中进行的一种称重尺度方法。在这里, 我们提出了一个训练老鼠的协议, 在一个以操作为基础的决策范式中, 老鼠选择更高的奖赏, 代价是等待十五年代的接收。

视频: Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学:

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

科研

神经科学

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

在鼠标的操作性感觉寻求

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

T-maze Forced Alternation and Left-right Discrimination Tasks for Assessing Working and Reference Memory in Mice

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in rodents. In our laboratory, we evaluated the two types of memory in more than 30 strains of genetically engineered mice using the automated version of this apparatus. Here, we present the modified T-maze apparatus operated by a computer with a video-tracking system and our protocols in a movie format. The T-maze apparatus consists of runways partitioned off by sliding doors that can automatically open downward, each with a start box, a T-shaped alley, two boxes with automatic pellet dispensers at one side of the box, and two L-shaped alleys. Each L-shaped alley is connected to the start box so that mice can return to the start box, which excludes the effects of experimenter handling on mouse behavior. This apparatus also has an advantage that in vivo microdialysis, in vivo electrophysiology, and optogenetics techniques can be performed during T-maze performance because the doors are designed to go down into the floor. In this movie article, we describe T-maze tasks using the automated apparatus and the T-maze performance of &alpha;-CaMKII+/- mice, which are reported to show working memory deficits in the eight-arm radial maze task. Our data indicated that &alpha;-CaMKII+/- mice showed a working memory deficit, but no impairment of reference memory, and are consistent with previous findings using the eight-arm radial maze task, which supports the validity of our protocol. In addition, our data indicate that mutants tended to exhibit reversal learning deficits, suggesting that &alpha;-CaMKII deficiency causes reduced behavioral flexibility. Thus, the T-maze test using the modified automatic apparatus is useful for assessing working and reference memory and behavioral flexibility in mice.

This article presents the protocol of T-maze tests using a modified automated apparatus for assessing the learning and memory functions in mice.

T-迷宫强迫交替和评估工作，并参考在小鼠记忆的左，右的歧视任务

Forced alternation and left-right discrimination tasks using the T-maze have been widely used to assess working and reference memory, respectively, in ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

一个多认知功能测试，并记录在啮齿动物的神经元活动的完全自动化和高度灵活的系统

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

抛光和钢筋，颅骨变薄窗口，为长期的小鼠脑成像

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

经颅磁刺激在线联合方法对经颅交流电刺激对原发性运动皮层的影响

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...